Solubility Tag Research Articles

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Functional Expression and Construction of a Self-Sufficient Cytochrome P450 Chimera for Efficient Steroidal C14α Hydroxylation in Escherichia coli.

C14-functionalized steroids enabled diverse biological activities in anti-gonadotropin and anticancer therapy. However, access to C14-functionalized steroids was impeded by the deficiency of chemical synthetic methods. Recently, several membrane-bound fungal cytochrome P450s (CYPs) have been identified with steroid C14α-hydroxylation activity. However, the lack of efficient heterologous overexpression strategy hampered their further characterization and molecular engineering. In the present study, sequences of fungi-derived CYP genes encoding putative 14α-hydroxylase were selected and bioinformatically analyzed. Substitution of the N-terminal hydrophobic helix by a soluble maltose binding protein tag significantly enhanced the soluble expression level in Escherichia coli. A novel CYP originated from Bipolaris oryzae was discovered with high steroidal C14α-hydroxylation activity when coupled with the redox partner CPRlun. A catalytically self-sufficient chimeric CYP-CPR was built by intramolecular fusion, and the electronic transfer rate was improved. A coenzyme NADPH regeneration system was finally constructed by the co-expression of glucose dehydrogenase. The developed soluble multi-enzyme cascade biotransformation system supported the selective C14α-hydroxylation toward progesterone with a final titer of 34.54 mg/L, the highest level achieved in E. coli-based heterologous expression system. This study provides insightful ideas on the functional expression of fungi-derived CYPs and promises an efficient C14α-hydroxylation system for steroidal drugs through protein engineering.

New insight into a simple high-yielding method for the production of fully folded and functional recombinant human CCL5

Chemokines are proteins important for a range of biological processes from cell-directed migration (chemotaxis) to cell activation and differentiation. Chemokine C-C ligand 5 (CCL5) is an important pro-inflammatory chemokine attracting immune cells towards inflammatory sites through interaction with its receptors CCR1/3/5. Recombinant production of large quantities of CCL5 in Escherichia coli is challenging due to formation of inclusion bodies which necessitates refolding, often leading to low recovery of biologically active protein. To combat this, we have developed a method for CCL5 production that utilises the purification of SUMO tagged CCL5 from E. coli SHuffle cells avoiding the need to reform disulfide bonds through inclusion body purification and yields high quantities of CCL5 (~ 25 mg/L). We demonstrated that the CCL5 produced was fully functional by assessing well-established cellular changes triggered by CCL5 binding to CCR5, including receptor phosphorylation and internalisation, intracellular signalling leading to calcium flux, as well as cell migration. Overall, we demonstrate that the use of solubility tags, SHuffle cells and low pH dialysis constitutes an approach that increases purification yields of active CCL5 with low endotoxin contamination for biological studies.

Improved production of class I phosphatidylinositol 4,5-bisphosphate 3-kinase

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.

Cloning and Direct Evolution of a Novel 7-O-Glycosyltransferase from Cucurbita moschata and Its Application in the Efficient Biocatalytic Synthesis of Luteolin-7-O-glucoside.

Luteolin-7-O-glucoside(L7G), a glycosylation product of luteolin, is present in a variety of foods, vegetables, and medicinal herbs and is commonly used in dietary supplements due to its health benefits. Meanwhile, luteolin-7-O-glucoside is an indicator component for the quality control of honeysuckle in the pharmacopoeia. However, its low content in plants has hindered its use in animal pharmacological studies and clinical practice. In this study, a novel 7-O-glycosyltransferase CmGT from Cucurbita moschata was cloned, which could efficiently convert luteolin into luteolin-7-O-glucoside under optimal conditions (40 °C and pH 8.5). To further improve the catalytic efficiency of CmGT, a 3D structure of CmGT was constructed, and directed evolution was performed. The mutant CmGT-S16A-T80W was obtained by using alanine scanning and iterative saturation mutagenesis. This mutant exhibited a kcat/Km value of 772 s-1·M-1, which was 3.16-fold of the wild-type enzyme CmGT. Finally, by introducing a soluble tag and UDPG synthesis pathway, the strain BXC was able to convert 1.25 g/L of luteolin into 1.91 g/L of luteolin-7-O-glucoside under optimal conditions, achieving a molar conversion rate of 96% and a space-time yield of 27.08 mg/L/h. This study provides an efficient method for the biosynthesis of luteolin-7-O-glucoside, which holds broad application prospects in the food and pharmaceutical industry.

Bacterial Production of CDKL5 Catalytic Domain: Insights in Aggregation, Internal Translation and Phosphorylation Patterns.

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in human brain development and functioning. Mutations in CDKL5, especially in its catalytic domain, cause a severe developmental condition named CDKL5 deficiency disorder. Nevertheless, molecular studies investigating the structural consequences of such mutations are still missing. The CDKL5 catalytic domain harbors different sites of post-translational modification, such as phosphorylations, but their role in catalytic activity, protein folding, and stability has not been entirely investigated. With this work, we describe the expression pattern of the CDKL5 catalytic domain in Escherichia coli demonstrating that it predominantly aggregates. However, the use of solubility tags, the lowering of the expression temperature, the manual codon optimization to overcome an internal translational start, and the incubation of the protein with K+ and MgATP allow the collection of a soluble catalytically active kinase. Interestingly, the resulting protein exhibits hypophosphorylation compared to its eukaryotic counterpart, proving that bacteria are a useful tool to achieve almost unmodified CDKL5. Posing questions about the CDKL5 autoactivation mechanism and the determinants for its stability, this research provides a valuable platform for comparative biophysical studies between bacterial and eukaryotic-expressed proteins, contributing to our understanding of neurodevelopmental disorders associated with CDKL5 dysfunction.

Rapid and reversible dissolution of biomolecular condensates using light-controlled recruitment of a solubility tag

Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function – dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Production of recombinant human epidermal growth factor fused with HaloTag protein and characterisation of its biological functions.

Epidermal growth factor (EGF) protein is a crucial biomolecule involved in regulating cell growth, proliferation, migration and differentiation, which is used in various therapeutic applications, such as wound healing and tissue regeneration. The production of recombinant EGF is essential for studying its biological function and for its clinical translation. However, EGF protein expressed in prokaryotic cells often occurs in inclusion bodies, and co-expression with soluble tag protein is an effective method to prepare recombinant EGF. In this study, we expressed recombinant human EGF (rhEGF) fused to a HaloTag (Halo-rhEGF) and a large portion of Halo-rhEGF was found in the soluble fraction. Cell growth assay showed that the purified Halo-rhEGF protein could promote the proliferation of fibroblasts (NIH 3T3) and epithelial cells (HaCaT), and significantly increased their viability. Phosphorylation of the intracellular signaling proteins, ERK1/2 and c-Jun, was stimulated by treatment with Halo-rhEGF and the expression levels of proteins regulating cell proliferation were significantly increased. RNA sequencing analysis revealed that rhEGF could increase the transcription of genes enriched in ribosome generation and cell proliferation. Moreover, Halo-rhEGF can be labelled by HaloTag ligand for fluorescence imaging and can be slowly released in tissue repair by binding to anion biomaterials. In conclusion, HaloTag is an efficient fusion tag for rhEGF protein expression, purification and controlled release, and Halo-rhEGF can promote the proliferation and viability of epithelial and fibroblast cells.

Protease-Driven Phase Separation of Elastin-Like Polypeptides.

Elastin-like polypeptides (ELPs) are a promising material platform for engineering stimuli-responsive biomaterials, as ELPs undergo phase separation above a tunable transition temperature. ELPs with phase behavior that is isothermally regulated by biological stimuli remain attractive for applications in biological systems. Herein, we report protease-driven phase separation of ELPs. Protease-responsive "cleavable" ELPs comprise a hydrophobic ELP block connected to a hydrophilic ELP block by a protease cleavage site linker. The hydrophilic ELP block acts as a solubility tag for the hydrophobic ELP block, creating a temperature window in which the cleavable ELP reactant is soluble and the proteolytically generated hydrophobic ELP block is insoluble. Within this temperature window, isothermal, protease-driven phase separation occurs when a critical concentration of hydrophobic cleavage product accumulates. Furthermore, protease-driven phase separation is generalizable to four compatible protease-cleavable ELP pairings. This work presents exciting opportunities to regulate ELP phase behavior in biological systems using proteases.

Scalable preparation of green C-terminal amidation peptide-synthesis TAGs and the optimized TAG-assisted liquid-phase synthesis of eptifibatide

Eptifibatide is a cycloheptapeptide with C-terminal amidation and head-to-tail disulfide bridging, which has been approved as an antithrombotic drug. Currently, both at the laboratory scale and industrial level, the synthesis of eptifibatide predominantly relies on solid-phase peptide synthesis (SPPS), which is not regarded as a green sustainable and cost-effective approach to prepare eptifibatide. Herein, we have investigated the strategy employing tag-assisted liquid-phase peptide synthesis (TAG-Assisted LPPS) technology that is scalable to multi-gram scale manufacturing for eptifibatide preparation. We successfully obtained soluble Rink-Amide TAGs by esterifying organophosphorus TAG-OH with Rink-Amide, thereby facilitating the transition from synthesizing C-terminal carboxyl peptides to C-terminal amidation peptides. The products obtained during the tag-assisted coupling and Fmoc deprotection of eptifibatide intermediates can be purified through precipitation processes, eliminating the need for complex chromatographic operations. Accordingly, we developed an optimized laboratory-scale process (300 mmol) for the preparation of TAG-Rink Amide TAG, which was subsequently upscaled successfully to a 50 mmol batch synthesis of eptifibatide. The subsequent work also explores the processes of peptide cleavage and disulfide bond oxidative cyclization in eptifibatide preparation.

Improved Expression of Aggregation-Prone Tau Proteins Using a Spidroin-Derived Solubility Tag

Tauopathies, a group of neurodegenerative disorders, are characterized by the abnormal aggregation of microtubule-associated Tau proteins in neurons and glial cells. The process of Tau proteins transitioning from soluble, intrinsically disordered monomers to disease-associated aggregates is still unclear. Investigating these molecular mechanisms requires the reconstitution of such processes in cellular and in vitro models using recombinant proteins at high purity and yield. However, the production of phase-separating or aggregation-prone recombinant proteins like Tau’s hydrophobic-rich domains or disease mutation-carrying variants on a large scale is highly challenging due to their limited solubility. To overcome this challenge, we have developed an improved strategy for expressing and purifying recombinant Tau proteins using the major ampullate spidroin-derived solubility tag (MaSp-NT*). This approach involves using NT* as a fusion tag to enhance the solubility and stability of expressed proteins by forming micelle-like particles within the cytosol of E. coli cells. We found that fusion with the NT* tag significantly increased the solubility and yield of highly hydrophobic and/or aggregation-prone Tau constructs. Our purification method for NT* fusion proteins yielded up to twenty-fold higher amounts than proteins purified using our novel tandem-tag (6xHis-SUMO-Tau-Heparin) purification system. This enhanced expression and yield were demonstrated with full-length Tau (hT40/Tau441), its particularly aggregation-prone repeat domain (Tau-MTBR), and Frontotemporal dementia (FTD)-associated mutant (Tau-P301L). These advancements offer promising avenues for the production of large quantities of Tau proteins suitable for in vitro experimental techniques such as nuclear magnetic resonance (NMR) spectroscopy without the need for a boiling step, bringing us closer to effective treatments for tauopathies.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Unexpected tobacco etch virus (TEV) protease cleavage of recombinant human proteins

The tobacco etch virus (TEV) protease is a commonly used reagent for removal of solubility and purification tags from recombinant proteins and is cited as being highly specific for its canonical cleavage site. Flexibility in some amino acids within this recognition sequence has been described in the literature but researchers generally assume few native human proteins will carry off-target sequences for TEV cleavage. We report here the aberrant cleavage of three human proteins with non-canonical TEV protease cleavage sites and identify broader sequence specificity rules that can be used to predict unwanted cleavage of recombinant proteins. Using these rules, 456 human proteins were identified that could be substrates for unwanted TEV protease cleavage.

Improvement of Carotenoids' Production by Increasing the Activity of Beta-Carotene Ketolase with Different Strategies.

Canthaxanthin is an important antioxidant with wide application prospects, and β-carotene ketolase is the key enzyme involved in the biosynthesis of canthaxanthin. However, the challenge for the soluble expression of β-carotene ketolase is that it hinders the large-scale production of carotenoids such as canthaxanthin and astaxanthin. Hence, this study employed several strategies aiming to improve the soluble expression of β-carotene ketolase and its activity, including selecting optimal expression vectors, screening induction temperatures, adding soluble expression tags, and adding a molecular chaperone. Results showed that all these strategies can improve the soluble expression and activity of β-carotene ketolase in Escherichia coli. In particular, the production of soluble β-carotene ketolase was increased 8 times, with a commercial molecular chaperon of pG-KJE8, leading to a 1.16-fold enhancement in the canthaxanthin production from β-carotene. Interestingly, pG-KJE8 could also enhance the soluble expression of β-carotene ketolase derived from eukaryotic microalgae. Further research showed that the production of canthaxanthin and echinenone was significantly improved by as many as 30.77 times when the pG-KJE8 was added, indicating the molecular chaperone performed differently among different β-carotene ketolase. This study not only laid a foundation for further research on the improvement of β-carotene ketolase activity but also provided new ideas for the improvement of carotenoid production.

A strategy for producing isotopically labeled peptides with antimicrobial activity or with short in vivo lifetime in Escherichia coli

AbstractEngineered Escherichia coli (E. coli) strains have been widely used to produce isotopically labeled peptides for NMR characterization on their structures and interactions. However, production of antimicrobial peptides (AMPs) by E. coli is still challenging, because AMPs are toxic to E. coli host and would lead to cell death after induction. On the other hand, expression of short peptides in E. coli host often encounter problems of the short in vivo lifetime of the peptides, which were rapidly degraded by endogenous enzymes during expression and purification steps. This report presents a practical method for overcoming these bottlenecks to enable E. coli to express AMPs and peptides that have short in vivo lifetime. This design uses the fusion of thioredoxin tags at both the N‐ and C‐termini of the target peptides. The steric effect of the large soluble tags at both ends of the peptide reduces peptide accessibility, thereby enhancing their in vivo stability and eliminating the toxicity associated with AMPs. The approach was validated using an AMP A3K/L7K‐LAH4 (K3K7) and a membrane fusion peptide (FP), which is a segment of the spike protein of SARS‐CoV‐2 and functions in fusing viral membranes and host cell membranes. Fusion expression of K3K7 with a thioredoxin tag only at the N‐terminal resulted in high toxicity to the host cells, leading to impaired cell growth and a failure to obtain expressed fusion protein. In contrast, the fusion proteins from both termini were successfully expressed and purified. In the case of the FP, the fusion of thioredoxin at both termini significantly enhanced its stability, protecting it from enzymatic degradation during expression and purification steps. On the contrary, the FP with thioredoxin fused only at the N‐terminal was found to be unstable in E. coli host strains. As stable isotope labeling on peptide is essentially important in NMR‐based structure and interaction studies, we also demonstrated that the developed approach enables efficient 15N labeling for NMR studies. This strategy may also be extended to produce other challenging peptides.

Discovery of l-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids

Beta-hydroxy non-standard amino acids (β-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for β-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of β-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineered Escherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.

Synthetic surfactant with a combined SP-B and SP-C analogue is efficient in rabbit models of adult and neonatal respiratory distress syndrome

Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.

Acrylic acid and DMSP lyases in the green algae Ulva

Green macroalgae of the genus Ulva contain dimethylsulfoniopropionate (DMSP) produced from methionine through a transamination pathway. DMSP is converted by DMSP lyase enzymes (DLs) into acrylic acid (AA), a high-value compound with a vast market as its esters have applications in the production of plastic additives, textiles, sealants, adhesives and surface coatings. The levels of AA produced by Ulva species, and the processes to extract it from this biomass, are currently poorly understood. In this study an analytical method was implemented for the simultaneous measurement of AA, DMSP and the pathway intermediates (4-methylthio-2-oxobutyrate, 4-methylthio-2-hydroxybutyrate, and 4-dimethylsulfonio-2-hydroxybutyrate) in Ulva biomass. The amounts of these compounds – with a focus on AA – were then quantified after processing the macroalgae with different chemical, thermal, and enzymatic treatments. AA was extracted in the range of 0.22–0.45 % of the algal dry weight. Sequential enzyme treatments showed no higher yields compared to individual enzymatic treatments or chemico-physical extractions, an encouraging result for the application of the treatments in industry. Codon-optimised synthetic genes for two candidate DLs from Ulva mutabilis were expressed and purified in E. coli with solubility tags, and the biochemical characteristics of these recombinant lyases were investigated. Both enzymes UM021_MBP and UM030_Halo7 had lower activity than their microalgal counterpart Alma1, with whom they share 33.9 % and 39.7 % similarity at amino acid level respectively. UM030_Halo7 appeared more active than UM021_MBP, with Km and Vm of 7.10 mM and 5.99 mM/min/mg protein respectively. UM030_Halo7 showed a preferred pH of 6.0, an optimal of temperature of 20 °C, was inhibited by NaCl concentration equal or higher than 0.5 M but not by H2O2 up to 50 μM. These results advance our understanding of AA biosynthesis in Ulva at the molecular level, and could be useful to implement processes for extracting AA from Ulva species within a biorefinery framework.

Production of bioactive recombinant monoclonal antibody fragment in periplasm of Escherichia coli expression system

The microbial expression system (Escherichia coli) is the most widely studied host for the production of biotherapeutic products, such as antibody fragments, single chain variable fragments and nanobodies. However, recombinant biotherapeutic proteins are often expressed as insoluble proteins, thereby limiting the utility of E. coli as expression system. To overcome this limitation, various strategies have been developed, such as changes at DNA level (codon optimization), fusion with soluble tags and variations in process parameters (temperature), and inducer concentration. However, there is no “one size fits all” strategy. The most commonly used approach involves induction at low temperature, as reducing the temperature during cultivation has been reported to increase bioactive protein production in E. coli. In this study, we examine the impact of various process parameters, such as temperature and inducer concentration, as well as, high plasmid copy number vector for achieving enhanced soluble expression of TNFα inhibitor Fab. An interaction amongst these parameters has been observed and their optimization has been demonstrated to result in expression of 30 ± 3 mg/L antibody fragment using E. coli. This case study illustrates how process optimization can contribute toward making biotherapeutics affordable.

Rational peptide design for regulating liquid–liquid phase separation on the basis of residue–residue contact energy

Since liquid–liquid phase separation (LLPS) of proteins is governed by their intrinsically disordered regions (IDRs), it can be controlled by LLPS-regulators that bind to the IDRs. The artificial design of LLPS-regulators based on this mechanism can be leveraged in biological and therapeutic applications. However, the fabrication of artificial LLPS-regulators remains challenging. Peptides are promising candidates for artificial LLPS-regulators because of their ability to potentially bind to IDRs complementarily. In this study, we provide a rational peptide design methodology for targeting IDRs based on residue–residue contact energy obtained using molecular dynamics (MD) simulations. This methodology provides rational peptide sequences that function as LLPS regulators. The peptides designed with the MD-based contact energy showed dissociation constants of 35–280 nM for the N-terminal IDR of the tumor suppressor p53, which are significantly lower than the dissociation constants of peptides designed with the conventional 3D structure-based energy, demonstrating the validity of the present peptide design methodology. Importantly, all of the designed peptides enhanced p53 droplet formation. The droplet-forming peptides were converted to droplet-deforming peptides by fusing maltose-binding protein (a soluble tag) to the designed peptides. Thus, the present peptide design methodology for targeting IDRs is useful for regulating droplet formation.